The field of this invention is optical alignment of capillaries for detection of luminescence of a solute in a capillary.
Microfluidics holds great promise in a number of areas associated with separations, reactions, chemical operations, analysis, sequencing and the like. For many of these areas, it is necessary to detect by optical means a signal emanating from the capillary. While the capillaries are quite small, usually having a width of less than about 200 xcexcm, compared to molecules and particles, including cells, the width is quite large. Therefore, as these entities pass through a detection zone, it is important that the light beam used for activation be properly directed to the capillary and that the detectors be properly aligned to detect the emitted light from the capillary.
In the systems being developed today, high throughput using low volumes of samples and reagents is of critical importance. Researchers need to rapidly perform and analyze large numbers of chemical and biological operations in a miniaturized, automated format. Current approaches involve the use of lab chips having arrays of capillary or microfluidic networks disposed in a substrate. Within each network, it is possible to independently perform a step, a series of steps or a complete analysis by manipulating small volumes of fluids to conduct chemical operations such as mixing, diluting, concentrating and separating reagents. These manipulations are accomplished through the use of high voltages, pneumatics, and the like.
When applying this technology in the context of microfluidic networks patterned into an array, one frequently wishes to compare results from one network to another. Unless the excitation light sources are providing the same level of irradiation in the same area of the relevant capillary channels, the results will generally not be comparable. Proper alignment of optical detection systems comprised of the excitation source is therefore a necessary requirement.
Further complicating matters is the fact that plastics are becoming the preferred substrate for lab chips. In one aspect, many plastics that are useful for lab chips have a tendency to autofluoresce when exposed to a light source. When light irradiates the capillary walls or plastic substrate in which the capillary is formed, there is a substantial increase in the fluorescent background which substantially increases the noise-to-channel signal ratio. Researchers must be able to reliably distinguish the signal related to the channel contents from the background autofluorescence of the plastic substrate. In another aspect, the channel walls in plastic lab chips are generally not orthogonal to the surface of the chip. Depending upon the incident angle and site of entry of the light source into the channel, the depth of irradiation and the volume irradiated will vary thereby increasing the need for precise and consistent positioning of the light source.
Another challenge related to plastic lab chips arises from the processes commonly used in their manufacture. Prevalent examples of such processes include hot embossing, injection molding, extrusion and the like. In hot embossing, a plastic film is heated and then stamped with microfluidic patterns. With injection molding, plastic is heated to a viscous consistency and then pressed into a specific mold, resulting in a chip having microfluidic patterns. With extrusion, plastic pellets are heated and then forced through a die creating either a single or multiple layer substrate film. This hot film can then be either embossed or run through a gravure roller system which imprints microfluidic patterns onto the heated substrate. Required by each of these processes is the heating of plastic so that it becomes pliable and capable of deformation. The dilemma relative to this heating is that as plastic cools, it shrinks in a non-uniform manner creating irregularities in the substrates and the patterns contained therein.
Current technology related to manufacturing techniques for plastic lab chips has overcome the shrinkage problem with regards to reproducibly generating microchannels having substantially similar profiles and dimensions. However, consistent uniformity with regards to the positioning of these microchannels within specific arrays or patterns has yet to be achieved. This variability is problematic at the microscale level, particularly when trying to align an optical detection system relative to the individual networks. In one critical aspect the optical detection system needs to be mechanically positioned relative to the specific detection zones in each network. In another aspect, the optical properties of plastic polymer chains become anisotropic after the aforementioned shrinkage, contributing to inconsistencies in background autofluorescence and other variables such as refractive indices. Therefore, having an approach to optimally position the irradiation source relative to the microfluidic channels becomes very important in order to minimize background noise and generate reproducible and quantifiable channel signals.
Finally, it is also desirable to have fairly simple optical systems that do not require expensive optical trains to identify the site in the channel to be irradiated.
U.S. Pat. Nos. 5,545,901 and 5,614,726 describe different ways to position excitation beams in a capillary channel.
Methods and apparatus are provided for determining the location of a sloped side wall of a microfluidic channel within a lab chip that is composed of a light transmitting substrate. The methods provide for scanning the lab chip with a light source in a plane that is normal to the microfluidic channel, measuring the resulting light produced at one or more edges of the chip, and correlating the resulting light to relative locations within the lab chip. An apparatus is provided which includes a light source, a carriage system for moving the light source relative to the lab chip, one or more scatter detectors, and a computer processor for analyzing the signals from the scatter detectors. Variations are provided where the methods can be used to align optical detection systems at analogous locations relative to similar channels.